10 1. Field of the Invention
The present invention provides novel compounds phosphoinositides and analogues tagged with stable or radioactive isotopes, novel methods for their preparation by syntheses, and novel key intermediates of synthesis; the novel methods of synthesis are applied also for the preparation of the phosphoinositides in non-labelled form.
2. Related Art
Several phosphoinositides of eukaryotic cells, including 1D-1-(1xe2x80x2-O-fattyacylxe2x80x2-2xe2x80x2-O-fattyacyl -sn-glycero-3xe2x80x2-phospho)-myo-inositol (phosphatidylinositol, PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4-P), and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) have been well known as metabolically vital lipid precursors of the intracellular second messengers 1D-myo-inositol-1,4,5-trisphosphate and 1,xe2x80x22xe2x80x2-diacyl-sn-glycerol (see Berridge, M. J. 1987, Annu. Rev. Biochem., 56: 59). Recently, the D-3-phosphorylated phosphoinositides, including the phosphatidylinositol 3-phosphate (PtdIns-3-P), phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P2), phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) have been encountered in eukaryotic cells (Whitman, M., et al., 1987, Biochem. J., 247: 165; Whitman, M., et al., 1988, Nature, 332: 644 ), and, recognized as intracellular messengers (Stephens, L., et al., 1993, J. Biol. Chem., 268: 17162; Duckworth, B. C. and Cantley, L. C. Lipid Second Messengersxe2x80x94Handbook of Lipid Research; Plenum Press: New York, N.Y. 1996, Vol 8, pp 125-175.). Thus the phosphoinositides, and their metabolites, regulate vital biological signaling, and as such, are important materials for research studies, diagnostics reagents, and biotechnology aids. The various enzyme systems involved in the signal transduction via the phosphoinositides, especially the phosphoinositide-specific lipases A, C and D, the phosphoinositide kinases, and phosphoinositide-phosphate phosphatases regulate vital metabolic and physiological processes including cell division, growth and apoptosis. Therefore, phosphoinositides and analogues are being studied for the development of new drug modalities for aberrant signaling including some types of cancer (Kozikowski, A. P. et al., 1993, U.S. Pat. No. 5,227,508).
The phosphoinositides are extremely minor components of plasma and nuclear membranes of cells. Small quantities of PtdIns, PtdIns-4-P, and PtdIns-4,5-P2 can be obtained from natural sources, such as bovine brains, but the D-3-phosphorylated types are not available. The isolated materials are mixtures of molecular species differing in the nature and proportion of the integral fattyacyl ester residues. Individual molecular species with specified fattyacyls or equivalent are required as biochemical research reagents and must be prepared by synthesis. The utility of biochemical research reagent is enhanced by tagging the molecule with isotope labels, including radioactive atom labels, and phosphoinositides with such labels are useful materials.
In the prior art, labelled phosphoinositides have been prepared from tritium labelled myo-inositol using the biochemical machinery of intact cells; alternatively, the biochemical reaction of 32P labelled ATP catalyzed by PtdIns kinase enzymes has been used to introduce an additional phosphate albeit with 32P into bovine brain derived phosphoinositides (see Stephens, L., et al., 1993, J. Biol. Chem., 268: 17162; Duckworth, B. C. and Cantley, L. C. Lipid Second Messengersxe2x80x94Handbook of Lipid Research; Plenum Press: New York, N.Y. 1996, Vol 8, pp 125-175). The routes are inefficent, limited to minute quantities, and in case of kinase enzymes are practical only in the very few laboratories with access to these enzymes. All labelled phosphoinositides so produced are mixtures of molecular species differing in the nature and proportions of the integral fattyacyl ester residues; individual molecular species must be prepared by synthesis, and appropriate synthetic methods are not available.
Only one chemical route has been described whereby a diether analogue of PtdIns-4,5-P2 labelled with tritium was obtained; herein, the tritium label was introduced in the alkyl-ether chain by metal catalyzed reduction of a Cxe2x80x94C double bond with tritium gas (Chen, J. and Prestwich, G. D., 1996, J. Labelled Compounds and Radiopharmaceuticals, 39: 251-258). The method is not applicable to phosphoinositides with (poly)unsaturated fattyacyls, and for labeling at other locations, particularly in the inositol and glycerol residues; overall the method is inadequate.
Several chemical syntheses of the phosphoinositides without isotope labels have been described (for example, Aneja, S. G., et al., 1997, Tetrahedron Lett., 38: 803; Bruzik, K. S. and Kubiak, R. J. 1995, Tetrahedron Lett., 36: 2415; Chen, J., et al., 1996, J. Org. Chem., 91: 6305; Desai, T., et al., 1996, Special Publicationxe2x80x94Royal Society of Chemistry, 180: 67; Gaffney, P. R. J. and Reese, C. B., 1997, Bioorg. Med. Chem. Lett., 7: 3171; Gou, D.-M. and Chen, C.-S., 1994, J. Chem. Soc., Chem. Commun., 2125; Grove, S. J. A., et al., 1997, J. Chem. Soc., Chem. Commun., 1635; Toker, A., et al., 1994, J. Biol. Chem., 269: 32358; Watanabe, Y., et al., 1994, Tetrahedron Lett., 35: 123; Watanabe, Y., et al., 1995, Tetrahedron, 51:8969; Watanabe, Y. and Nakatomi, M., 1998, Tetrahedron Lett., 39: 1583). Most of these syntheses are applicable only to analogues with saturated fattyacyls at sn-glycero-1xe2x80x2,2xe2x80x2-O locations. The two most recent (Gaffney and Reese, 1997; Watanabe and Nakatomi, 1998) address the synthesis of phosphoinositides with unsaturated fattyacyls. As mentioned, these methods are not suitable for labelling phosphoinositides, and have not been so applied.
The present invention pertains to phosphoinositides and novel analogues tagged with stable or radioactive isotopes, novel methods for their preparation by syntheses, and novel key intermediates of synthesis. The analogues include but are not limited to structural and stereochemical isomers of the cellular phosphoinositides, the corresponding thiophosphates and phosphonates, and the radyl and sphingo type inositolphospholipids. The novel labelled compounds are closely related to the cellular phosphoinositides shown in the generalized structure below. 
The labels are provided preferably as deuterium and tritium isotopes of hydrogen, and radioactive isotopes of phosphorus and sulphur, but are not limited to these atoms. Labels are located at selected positions in the lipid or the inositol(phosphate) residues of phosphoinositide structure and are variously in the fattyacyl or alkyl chain(s), the glycero residue, the inositol residue, phosphate, thiophosphate, phosphonate or equivalent group(s). Labels are introduced at selected positions by way of novel synthons which carry temporary protecting groups at positions other than the label site, and this site consequently is amenable to selective labeling. The design and synthesis of these temporarily protected synthons is an critical element of the present invention. The novel methods of preparation are multi-step syntheses and comprise assembly of the complete phosphoinositide skeleton from phosphatidyl and inositol(phosphate) synthons. The isotope atoms are introduced at relatively late stage during synthesis of these aforementioned synthon types and assembly of the phosphoinositide skeleton from the synthons. The late stage option is desirable, particularly for radioactive isotope labels because it minimizes handling of radioactive materials and waste disposal, and thus is beneficial for personnel and environmental safety. Although the emphasis is on labelled materials and methods for the preparation thereof, the novel methods of synthesis are shown to be suitable and valid for phosphoinositides with the normal stable isotope compositions based on 1H, 31P, and 32S atoms. The chemistry and protocols for products with or without isotope labels are identical and hence synthesis of either type is sufficient validation of the novel methodology. Individual molecular species of the phosphoinositides, with or without isotope labels, and their analogues and metabolites are useful as research biochemicals, diagnostic reagents, biotechnology process aids, and models for the development of drugs for treatment of aberrant signaling.
The isotope atom label may be located in the fattyacyl, glycerol, inositol and phosphate residues of the phosphoinositide structure, at specified single positions, or at multiple sites; all provide useful reagents. The labelled phosphoinositides can be monitored in the intact state or after biological interactions and transformations. These transformations can result in enzyme catalyzed fragmentation or addition of more groups such as phosphates. Several enzyme and effector protein systems have been characterized which interact with the phosphoinositides in the signaling cascades and metabolic pathways. The phosphoinositide PtdIns-4,5-P2 functions as the preferred substrate of the PI-PLC (Lee, S. B. and Rhee, S. G. 1995, Curr. Opin. Cell Biol., 183) and the phosphoinositide 3-kinase (PtdIns 3-kinase) enzyme families (Duckworth, B. C. and Cantley, L. C., 1996, loc cit.), and, as allosteric activating factor of cellular regulatory proteins with and without pleckstrin homology (PH) domains (Terui, T., et al, 1994, J. Biol. Chem., 269: 28130).
The action of PI-PLC on PtdIns-4,5-P2 causes hydrolysis to the two intracellular second messengers 1D-myo-inositol-1,4,5-trisphosphate and 1xe2x80x2,2xe2x80x2-diacyl-sn-glycerol (Berridge, M. J., 1993, Nature, 361: 315). Phospholipase A2 specifically releases the fattyacyl from the sn-glycero-2xe2x80x2- position with concomitant formation of the corresponding lyso-phosphoinositide. PtdIns kinases (e.g., PtdIns 3-kinase) introduce phosphate groups into phosphoinositides and the PtdIns-phosphate phosphatases (e.g., PTEN), remove phosphate groups from inositol positions specific to each enzyme. Tritium labelled phosphoinositides are generally useful for monitoring such transformations, and deuterium labelled analogues are useful for defining metabolites in vivo in human subjects by analyses using (HPLC coupled to) mass spectrometry.
Several phosphoinositides with 1xe2x80x2,2xe2x80x2-dipalmitoyl-sn-glycero lipid residue have become available recently. These synthetic analogues have contributed significantly to the current state of knowledge about the functions of the phosphoinositides as signal mediators, and have become established as important biochemical reagents. Labelled analogues of these, the 1xe2x80x2-saturated fattyacyl-2xe2x80x2-unsaturated fattyacyl-sn-glycero lipid residue types akin to the cellular phosphoinositides, and, analogues including structural and stereochemical isomers are required as research and diagnostic reagents. The present invention is concerned with all these materials, methods for their preparation by synthesis, and important intermediates in the synthetic methods.
As mentioned, the prior art methods are not suitable for preparing individual molecular species of labelled phosphoinositides, and have not been so applied. The novel methods of synthesis of the present invention are designed specifically for labelled phosphoinositides, and additionally provide concise syntheses of the non-labelled species; the latter are useful research reagents in their own right.